Her2 is a receptor tyrosine kinase and cellular proto-oncogene that plays a major role in breast and other human cancers. Her2 is a member of the ErbB - epidermal growth factor receptor (EGFR) family. Under physiologic conditions, Her2 functions by heterodimerizing with EGFR, Her3 or Her4 and then activating its tyrosine kinase. We developed a reconstituted in vitro system to study the heterodimerization and enzyme activation of Her2. We will study the structure of Her2 heterodimers using two mass spectrometry (MS) approaches, termed chemical footprinting MS, and we will combine this with molecular dynamics simulations to obtain atom level interpretations of the MS results. Conclusions from MS and molecular dynamics will be functionally tested using Fluorescence Resonance Energy Transfer (FRET) experiments and mutagenesis experiments in cell lines. This proposal will produce a sustained, powerful influence by producing a number of scientific firsts, chief among them is the first structural study of a Her2-Her3 heterodimer. Her2-Her3 heterodimers form a very potent signaling pair and it is has been shown that knocking down Her3 in Her2- positive breast cancer cell lines reverses oncogenic transformation. Achieving the aims of this project will advance scientific knowledge by providing new structural and functional information on Her2-Her3 and EGFR-Her2 heterodimers. Achieving the aims of this project will also advance clinical practice by identifying which Her2 and Her3 cancer associated mutations likely drive cancer cell growth and are sensitive to Her2 targeted drugs. This information will identify new patients who are likely to benefit from Her2 targeted drugs and will provide the key scientific findings to support a future breast cancer clinical trial.